RU2554746C1 - Method of obtaining overall fraction of extracellular nucleic acids from blood - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: method includes collecting blood in an anticoagulant solution, adding an equal volume of an elution buffer containing 1 M NaCl, 0.2 M NaHCO₃ (pH 9.3), 20 mM EDTA or a buffer containing 1 M NaCl, 20 mM EDTA. The obtained mixture is incubated at room temperature for 4-6 minutes while stirring, followed by separating the collected blood into plasma and a blood cell fraction by centrifuging and collecting the supernatant fluid, from which the overall fraction of extracellular nucleic acids (exNA) is extracted.

EFFECT: use of the invention shortens the duration of obtaining an overall fraction of exNA, increases the output of blood exNA, increases the sensitivity of detecting market NA, which are specific for cancerous diseases and pregnancy pathologies.

3 cl, 3 tbl, 4 ex

Description

The invention relates to the field of diagnostic medicine, namely to molecular genetic diagnostics, and is aimed at increasing the diagnostic efficiency of analytical systems based on the use of circulating extracellular nucleic acids (freely circulating in plasma and associated with the surface of blood cells).

The urgent task of diagnostic medicine is the timely and accurate identification of specific nucleic acid sequences associated with various diseases in patients. Particularly intensive research is underway in the development of tests based on the analysis of circulating extracellular blood nucleic acids (extracellular DNA), since blood is in direct contact with all organs / tissues, and a number of processes, such as pregnancy, impaired cell and tissue functions, and the development of tumors are accompanied by a change in composition circulating blood nucleic acids even at the very early stages of the development of these processes, in particular, when a tumor is difficult / impossible to localize by physical methods (1).

It was previously shown that the use of nucleic acids (sccNAs) associated with the surface of blood cells in diagnostics of vnNAs, along with widely used vnNAs that freely circulate in blood plasma (2), can significantly increase the sensitivity of diagnostic systems (3).

A known method for the isolation of vDNA associated with the surface of blood cells (4), which includes the following stages: collection of blood by gravity into tubes with phosphate buffer (10 mm) and EDTA (50 mm EDTA pH 7.5); separation of blood into plasma and cell fraction by centrifugation (20 min at 400 g); separation of the cellular fraction of blood into red blood cells and white blood cells; obtaining DNA bound to the surface of red blood cells and DNA bound to the surface of leukocytes by incubating cells with a phosphate eluting buffer (5 min incubation followed by precipitation of the cells by centrifugation for 20 min at 400 g); obtaining vDNA from the erythrocytatra and leukocyte fractions by incubating cells with a trypsin solution and then a trypsin inhibitor (5 min incubation with a trypsin solution, adding a trypsin inhibitor, stirring the solution, pelleting cells by centrifugation for 20 min at 400 g). VnDNA was isolated from blood plasma and the resulting eluates from the surface of blood cells using fine glass.

The disadvantages of this method are the laborious procedure for obtaining fractions containing vnDNA blood; as well as the use of trypsin in the process of obtaining vDNA, which requires careful implementation of protocols and monitoring the activity of the drug.

A known method for the isolation of vRNA associated with the surface of blood cells (5), which includes the following stages: collecting blood by gravity into tubes with phosphate buffer (10 mm) and EDTA (50 mMEDTA pH 7.5); separation of blood into plasma and cell fraction by centrifugation (20 min at 400 g); obtaining vnRNA of blood cells by incubating cells with a phosphate eluting buffer (5 min incubation, followed by centrifugation for 20 min at 400 g); obtaining vnRNA of blood cells by incubating cells with a trypsin solution and then a trypsin inhibitor (5 min incubation with a trypsin solution, adding a trypsin inhibitor, mixing and sedimenting cells by centrifugation for 20 min at 400 g). Isolation of vRNA from plasma was performed on glass fiber filters.

This method is characterized by the previously mentioned drawbacks, the total time for obtaining fractions containing vnRNA is 2 hours 42 minutes (time is calculated for processing 2 blood samples with a volume of 8 ml), the method cannot be automated.

Closest to the claimed method, the prototype, is a method for isolating vDNA from blood, which includes the following stages: collecting blood into vakuteyner; separation of blood into plasma and cell fraction by centrifugation (20 min at 400 g); obtaining vDNA as a result of sequential treatment of blood cells with phosphate eluting buffer (5 min incubation followed by centrifugation for 20 min at 400 g); then treatment of blood cells with trypsin solution (5 min incubation with trypsin solution, addition of trypsin inhibitor, mixing and sedimentation of cells by centrifugation for 20 min at 400 g). Blood vnDNA from the obtained supernatants was isolated on fiberglass filters using the Blood DNA isolation kit (BioSilica Ltd, Russia) (6).

The disadvantages of the method are the complexity and duration of obtaining fractions containing vnDNA blood: the total time to obtain fractions containing vnDNA is 2 hours 42 minutes. In addition, the known method requires constant monitoring of the effectiveness of the protease treatment. The method contains many stages, requires constant fixing of volumes, it is extremely difficult to automate. In this case, two fractions of vDNA are analyzed: separately, vDNA of blood plasma and separately vDNA from the cell fraction of blood, which doubles the cost of isolation of vDNA and PCR analysis of samples.

The objective of the invention is to develop a simple, fast and reproducible method for obtaining the total fraction of blood vnNA containing both vnNA from blood plasma and vnNA from the cellular fraction of blood for subsequent use of vnNA as a diagnostic material.

Effect: simplify, reduce the duration of the method and increase the yield of cnnc.

The problem is achieved by the proposed method, which consists in the following.

Blood collection is performed using a vacutainer or other blood collection and preservation tubes (e.g., Cell-Free DNA BCT, Streck, USA). An equal volume of an elution buffer (DDB) containing 1 M NaCl, 0.2 M NaHCO ₃ (pH 9.3), 20 mM EDTA or a buffer containing 1 M NaCl, 20 mM EDTA was added to an aliquot of blood, the mixture was incubated at room temperature for 4-6 minutes, gently mixing. Next, the mixture of blood and the DDB elution buffer is separated into the supernatant and the cell fraction by double centrifugation (5 min at 1000 g and 5 min at 1200 g). The resulting supernatant contains the total cfNA fraction, including plasma cfNA and cfNA of the cell fraction. Further, the cnNA is isolated from the supernatant by any suitable method for isolating nucleic acids, namely: by a method based on phenol-chloroform extraction; guanidine-phenolic method; by nucleic acid extraction using fine glass or glass fiber filters in the presence of chaotropic salts. Quantitative analysis of blood vnNA is also carried out by any known method for studying nucleic acids, such as: quantitative PCR and RT-PCR; methyl-specific quantitative PCR, a method for measuring the concentration of NK using fluorescent dyes (Hoechst 32258, SYBR Green II, DAPI, etc.).

The defining difference of the proposed method compared to the prototype is that an equal volume of an elution buffer containing 1 M NaCl, 0.2 M NaHCO ₃ (pH 9.3), 20 mM EDTA or a buffer containing 1 M NaCl is added to the sample of blood under study. , 20 mM EDTA, incubate the mixture at room temperature for 4-6 minutes with stirring, which provides a total fraction containing vnNA from blood plasma and vnNA from the blood cell fraction, which in turn allows to simplify the method, reduce its duration and increase nucleic acid output rial, which allows to increase the detection sensitivity of the marker DNA and RNA sequences.

The proposed method has the following advantages compared to the prototype:

- provides the lysis of a smaller number of blood cells than the prototype, and is resistant to deviations from the regulation by no more than 20%, which makes it suitable for use in clinical laboratories to perform routine studies and allows you to automate the process;

- allows you to reduce the time to obtain the total cfnV fraction by more than 4 times (from 2 hours 42 minutes to 34 minutes);

- allows you to almost 4 times reduce the total volume of the obtained fraction (from 44.5 ml to 12 ml), which leads to a decrease in the complexity and cost of reagents for the extraction of NK, which in turn reduces the cost of tests based on the use of blood VNA.

The invention is illustrated by the following examples of specific performance of the method.

Example 1

In the group of healthy donors (n = 10), the total fraction of vDNA was obtained by the prototype method (6) and the proposed method. All healthy volunteer donors were sampled with a vakuteyner with an anticoagulant. The collected blood was divided into 2 equal parts, one part was processed in three stages according to the prototype protocol, and the remaining part was processed in one stage by the claimed method: an equal volume of elution buffer (DDB) containing 1 M NaCl, 0.2 M was added to an aliquot of blood NaHCO ₃ (pH 9.3), 20 mM EDTA and the mixture was incubated at room temperature for 4 minutes, gently stirring, then the mixture of blood and DDB elution buffer was separated into supernatant and cell fraction by double centrifugation (5 min at 1000 g and 5 minutes at 1200 g), and from the resulting soup vnDNA rnatant was isolated using fine glass in the presence of chaotropic salts (4). DNA concentration was determined using TaqMan-PCR on LINE-1 repeats (7).

The results of the study are presented in Table 1. As can be seen from table 1, the use of the proposed method for obtaining the total fraction of blood vnDNA allows to increase the vnDNA yield by an average of 20.07%. At the same time, saving time is 2 hours 8 minutes.

This example illustrates the possibility of using the developed method in clinical laboratories using in-line methods with high reproducibility.

Example 2

In the group of patients with lung cancer (n = 10), the total fraction of vDNA obtained using the prototype method (6) and the proposed method was isolated. All people taking the study had blood taken with the help of a vacutainer. The collected blood was divided into 2 equal parts, one part was processed in three stages according to the prototype protocol, and the second part was processed in one stage using the proposed method, namely: an equal volume of elution buffer (DDB) containing 1 M was added to an aliquot of blood NaCl, 20 mm EDTA and incubated the mixture at room temperature for 6 minutes, gently mixing, then the mixture of blood and the DDB elution buffer was separated into supernatant and cell fraction by double centrifugation (5 min at 1000 g and 5 minutes at 1200 g), and from the floor ennogo supernatant extracellular DNA isolated using the kits BioSilika (Biosilika Ltd., Russia). The concentration of tumor-specific DNA was determined using methyl-specific SYBR-Green PCR on the methylated fragment of the RARbeta2 gene (8).

Table 2 shows the concentration of the methylated form of the RARβ2 gene in the composition of vDNA in the blood of patients with lung cancer, where Protocol 1 is the prototype method. Protocol 2 - the proposed method.

As can be seen from table 2, the use of the proposed method for obtaining the total fraction of vDNA from the blood of cancer patients can significantly increase the yield of tumor-specific vDNA by an average of 58%, while the time spent on the proposed method is reduced by more than 4 times (from 2 hours 42 minutes to 34 minutes).

Example 3

In the group of pregnant RhD-negative women waiting for an RhD-positive child (n = 20), a comparative study of the concentrations of the total fraction of vDNA and the Rh genetic marker (RHD) in samples obtained using the prototype method (6) and the proposed the method is similar to example 1. All patients were taken blood using a vacutainer. The collected blood was divided into 2 equal parts, one part was processed according to the prototype protocol to the stage of using eluting buffers (only blood plasma samples were obtained), and the second part was processed using the proposed method analogously to example 1, while the vDNA from the obtained supernatant was isolated using automatic QIAsymphony stations (Qiagen, USA). DNA concentrations were investigated using TaqMan-PCR for the GAPDH household gene fragment and RHD genetic marker for Rhesus affection (9) (Table 3).

As can be seen from table 3, the use of the proposed method allows an average of 6.5 times increase the concentration of vDNA. Moreover, the concentration of fetal DNA when using the proposed method increases by an average of 18%. This fact can be very important in the analysis of fetal DNA in women with early pregnancy, when the concentration of fetal DNA is very low. Using the proposed method of blood processing may allow RhD typing of the fetus in earlier pregnancy.

Example 4

In groups of patients with lung cancer (n = 10) and healthy donors (n = 10), the level of expression of circulating microRNAs (siRNAs) in blood was studied using siRNAs-21 and 155, the level of expression of which increases with the development of oncopathology (10). Obtaining fractions of vnRNA was carried out using the prototype method (6) and the proposed method. All people participating in the study had blood taken using a vacutainer. The collected blood was divided into 2 equal parts, one part was processed in three stages according to the prototype protocol, and the second part was processed in one stage as in example 1, while vnRNA was isolated from the obtained supernatant using guanidine phenolic extraction (11). The level of target siRNA expression was determined using quantitative RT-PCR (12). It was shown that the use of the proposed method for obtaining the total fraction of blood vnRNA allows to increase the yield of vnRNA targets by an average of 32%.

Using the proposed method will simplify and reduce the duration of the procedure, as well as increase the amount of diagnostic material, which will increase the sensitivity of detection of marker NK specific for cancer and pregnancy pathologies.

Information sources

1. Jung K., Fleischhacker M Rabien A. Cell-free DNA in the blood as a solid tumor biomarker-a critical appraisal of the literature // Clin Chim Acta. - 2010 .-- V. 411.P. 1611-1624.

2. Pinzani P., Salvianti F., Pazzagli M. et al. Circulating nucleic acids in cancer and pregnancy. // Methods. - 2010 .-- V. 50 (4). P. 302-307.

3. Rykova E.Y., Morozkin E.S., Ponomaryova A.A. et al. Cell-free and cell-bound circulating nucleic acid complexes: mechanisms of generation, concentration and content // Expert Opin Biol Ther. - 2012. - V. 12. P. 141-153.

4. Skvortsova T.E., Rykova E.Y., Tamkovich S.N., et al. Cell-free and cell-bound circulating DNA in breast tumors: DNA quantification and analysis of tumor-related gene methylation // Br J Cancer. - 2006. - V. 94.P. 1492-1495.

5. Rykova E.Yu., Skvortsova T.E., Hoffman A.L. et al. // Circulating extracellular DNA and blood RNA in the diagnosis of breast cancer. Biomedical chemistry. 2008, Volume 54, Issue 1, pp. 94-103.

6. Laktionov P.P., Tamkovich S.N., Rykova E.Yu. et al. A method for early diagnosis and monitoring of cancer. Patent RU 2251696 C2, publ. 05/10/2005

7. Morozkin E.S., Babochkina T.I., Vlassov V.V., Laktionov P.P. The effect of protein transport inhibitors on the production of extracellular DNA // Ann N Y Acad Sci. - 2008. - V. 1137. - P. 31-35.

8. Ponomaryova A.A., Rykova E.Y., Cherdyntseva N.V. et al. / RARβ2 gene methylation level in the circulating DNA from lung cancer patient blood // Eur J Cancer Prev. - 2011 V. - 20, N. 6. - P. 453-455.

9. Clausen F.B., Jakobsen T.R., Rieneck K. et al. Pre-analytical conditions in non-invasive prenatal testing of cell-free fetal RHD // PLoS One. - 2013 .-- V. 8 (10). e76990.

10. Jones K., Nourse J.P., Keane C. et al. Plasma microRNA are disease response biomarkers in classical Hodgkin lymphoma // Clin Cancer Res. - 2014. - V. 20. - P. 253-64.

11. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction // Anal Biochem. - 1987. - V. 162. - P. 156-159.

12. Chen S., Ridzon D.A., Broomer A.J. et al. Real-time quantification of microRNAs by stem-loop RT-PCR // Nucleic Acids Res. - 2005. - V. 33. - P. e179.

Claims (3)

1. A method for isolating the total fraction of extracellular nucleic acids from blood (vnNA), including collecting blood in an anticoagulant solution, separating the collected blood into plasma and the blood cell fraction by centrifugation, isolating vnNA from the resulting supernatant, followed by measuring the concentration of vnNA, characterized in that to the blood sample, before dividing it into fractions, add an equal volume of an elution buffer containing 1 M NaCl, 0.2 M NaHCO ₃ (pH 9.3), 20 mM EDTA or a buffer containing 1 M NaCl, 20 mM EDTA the mixture is incubated When room temperature for 4-6 minutes with stirring. 2. The method according to p. 1, characterized in that the cDNA is isolated from the supernatant by a method selected from: extraction with phenol-chloroform; Guanidine-phenol extraction; Isolation using fine glass or fiberglass filters in the presence of chaotropic salts. 3. The method according to p. 1, characterized in that the measurement of the concentration of cnNA is carried out by a method selected from: quantitative PCR and RT-PCR, methyl-specific quantitative PCR, a method for measuring the concentration of NK using fluorescent dyes.